TY - JOUR
T1 - Prefixation of neutrophils for neutrophil antibody testing
AU - Shim, Ye Jee
AU - Won, Dong
PY - 2014/3/1
Y1 - 2014/3/1
N2 - Objective: The granulocyte immunofluorescence test based on flow cytometry is still the most commonly used method for detecting antineutrophil antibodies. We questioned whether neutrophil prefixation is necessary for this test. Methods: Three pretreatment methods using paraformaldehyde were compared: unfixed, short-fixed (3 minutes 40 seconds), and long-fixed (10 minutes) leukocytes. The mean fluorescence intensity (MFI) of a test sample was divided by the average of negative controls, and this MFI ratio of gated neutrophils or lymphocytes was assessed for its discriminatory value based on the pretreatment method used. Results: In neutrophil antibody positive (Ab+) sera (n=7), the MFI ratio of unfixed neutrophils (1.97) was higher than that of short- (1.60, P =0.004) or long- (1.71, P =0.203) fixed neutrophils. In HLA Ab+ sera (n=10), the MFI ratio of short-fixed lymphocytes (8.85) was higher than that of unfixed (6.52, P =0.120) or long-fixed (3.86, P =0.055) lymphocytes. In antinuclear Ab+ sera (n=4), the MFI ratio of unfixed neutrophils (2.04) was higher than that of short- (1.23, P =0.047) or long- (1.14, P =0.044) fixed neutrophils. Conclusion: The benefit of a prefixation step depends on the diagnostic purpose. Unfixed neutrophils are more sensitive, but the prefixed neutrophils are more specific. For the diagnosis of autoimmune neutropenia of childhood, prefixation can be omitted to enhance sensitivity.
AB - Objective: The granulocyte immunofluorescence test based on flow cytometry is still the most commonly used method for detecting antineutrophil antibodies. We questioned whether neutrophil prefixation is necessary for this test. Methods: Three pretreatment methods using paraformaldehyde were compared: unfixed, short-fixed (3 minutes 40 seconds), and long-fixed (10 minutes) leukocytes. The mean fluorescence intensity (MFI) of a test sample was divided by the average of negative controls, and this MFI ratio of gated neutrophils or lymphocytes was assessed for its discriminatory value based on the pretreatment method used. Results: In neutrophil antibody positive (Ab+) sera (n=7), the MFI ratio of unfixed neutrophils (1.97) was higher than that of short- (1.60, P =0.004) or long- (1.71, P =0.203) fixed neutrophils. In HLA Ab+ sera (n=10), the MFI ratio of short-fixed lymphocytes (8.85) was higher than that of unfixed (6.52, P =0.120) or long-fixed (3.86, P =0.055) lymphocytes. In antinuclear Ab+ sera (n=4), the MFI ratio of unfixed neutrophils (2.04) was higher than that of short- (1.23, P =0.047) or long- (1.14, P =0.044) fixed neutrophils. Conclusion: The benefit of a prefixation step depends on the diagnostic purpose. Unfixed neutrophils are more sensitive, but the prefixed neutrophils are more specific. For the diagnosis of autoimmune neutropenia of childhood, prefixation can be omitted to enhance sensitivity.
KW - Flow cytometry
KW - Neutrophil antibody
KW - Prefixation
UR - http://www.scopus.com/inward/record.url?scp=84902080659&partnerID=8YFLogxK
U2 - 10.1309/LMG76BSOGJ7ICRFM
DO - 10.1309/LMG76BSOGJ7ICRFM
M3 - Article
C2 - 24868992
AN - SCOPUS:84902080659
SN - 0007-5027
VL - 45
SP - 120
EP - 131
JO - Laboratory Medicine
JF - Laboratory Medicine
IS - 2
ER -